弓形虫GJS株致密颗粒6基因真核表达质粒的构建及在BHK-21细胞中的表达

根据GenBank数据库中弓形虫RH株GRA6基因序列设计1对引物,采用PCR技术从弓形虫GJS株基因组DNA中扩增GRA6基因,导入真核表达载体pcDNA3.1/CT-GFP-TOPO,转化大肠杆菌DH5α,经酶切、PCR及测序鉴定,将重组质粒转染BHK-21细胞。GFP标签证明质粒DNA成功转染到细胞中并得以表达,通过Western-blot分析,细胞裂解液中有一条约52 ku的条带,可被山羊抗弓形虫超免疫血清识别,大小与预测值相符。表明真核表达质粒pcDNA3.1/CT-GFP-TOPO-GRA6中的GRA6基因在BHK-21细胞中获得表达且表达产物具有抗原性。     

刚地弓形虫GJS株微线体蛋白3基因真核表达质粒的构建及在BHK-21细胞中的表达

采用PCR技术从刚地弓形虫GJS株基因组DNA中扩增微线体蛋白3(MIC3)基因,并克隆到pMD18-T载体,经PCR、酶切及测序鉴定后,阳性重组质粒酶切并亚克隆到真核表达载体pcDNA3.1(+)后进行PCR、酶切及测序鉴定.将重组质粒转染BHK-21细胞.间接免疫荧光染色证明,质粒DNA成功转染到细胞中并得以表达.Western-blot分析证实,细胞裂解液及上清样品中有1条约39.2 ku的条带,可被山羊抗刚地弓形虫超免疫血清所识别,大小与预测值相符.表明,真核表达质粒pcDNA3-MIC3中的MIC3基因在BHK-21细胞中获得表达且表达产物具有抗原性.     

旋毛虫Ts43基因真核表达载体的构建及其在Vero E6细胞中的表达

利用RT-PCR技术从黑龙江省猪源旋毛虫获得了Ts43基因,并克隆入pcDNA3.1-CT-GFP真核表达载体中构建重组质粒,用该重组质粒在脂质体介导下转染Vero E6细胞,GFP标签证明质粒DNA成功转染到细胞中并得以表达,通过Western-blot分析,细胞裂解液样品中有1条约66 ku的条带,可被小鼠旋毛虫阳性血清所识别,与预计大小一致,说明,真核表达载体pcDNA3.1-CT-GFP中的Ts43基因在VeroE6细胞中获得了表达,表达产物具有抗原性。     

野鸭源新城疫病毒V蛋白基因在DF-1细胞中的表达及V蛋白的亚细胞定位

为研究野鸭新城疫病毒V蛋白基因在真核细胞中的表达及V蛋白的亚细胞定位,将新城疫病毒V基因片段与pcDNA3.1载体连接,构建pcDNA3.1-V重组表达质粒,经脂质体转染DF-1细胞后,用Western-blot验证该基因在DF-1细胞中的表达;用激光共聚焦显微成像技术确定V蛋白在真核细胞中的分布及亚细胞定位。结果显示,重组质粒pcDNA3.1-V在外源真核细胞DF-1中获得了表达,表达蛋白主要聚集于细胞质。研究结果为进行野鸭源新城疫病毒V蛋白的功能研究奠定了基础。     

IFN-λ1作为乙型脑炎病毒DNA疫苗佐剂的免疫效应研究

为了评估IFN-λ1作为免疫佐剂能否提高乙型脑炎病毒DNA疫苗的免疫效应,利用RT-PCR方法扩增猪源IFN-λ1基因,构建重组真核表达载体pVAX1-IFN-λ1,通过Western-blot和间接免疫荧光试验鉴定免疫效果。结果表明,转染pVAX1-IFN-λ1重组质粒的BHK21细胞能够正确表达IFN-λ1;将重组IFN-λ1与p VAX1-prME DNA疫苗免疫小鼠后,与对照组p VAX1-prME相比,IFN-λ1可以诱导小鼠产生乙型脑炎病毒EⅢ蛋白特异性抗体水平、乙脑病毒特异性中和抗体效价和淋巴细胞增殖水平显著提高。结论,重组猪源IFN-λ1作为乙型脑炎病毒DNA疫苗的分子免疫佐剂能够在小鼠动物模型上提高乙型脑炎病毒DNA疫苗的免疫效应。     

携带禽流感病毒DNA疫苗的减毒鼠伤寒沙门菌的免疫效力

将从重组质粒pVAX1-HA中扩增的HA基因和从pVAX1-HA-CpG中切出的HA-CpG基因分别克隆入pcDNA3.1(+)载体中,获得重组质粒pcDNA3.1-HA和pcDNA3.1-HA-CpG。将上述重组质粒分别转染COS-7细胞,经间接免疫荧光试验检测发现HA基因可在COS-7细胞中表达。将重组质粒转入减毒鼠伤寒沙门菌SL7207,构建成携带DNA疫苗的重组沙门菌SL7207(pcDNA3.1-HA)和SL7207(pcDNA3.1-HA-CpG)。将重组菌分别以5×109CFU口服免疫1日龄鸡2次,重组菌免疫组产生的小肠黏膜抗体效价与空载体组及油乳剂灭活疫苗组间存在显著差异。攻毒免疫保护结果表明,SL7207(pcDNA3.1-HA-CpG)免疫组与空载体组之间存在显著差异,而SL7207(pcDNA3.1-HA)免疫组则与空载体组间无显著差异,说明CpG可增强DNA疫苗的免疫效果。     

Ea3-1E与mChIL-15融合基因的构建及其真核表达质粒的免疫效果

应用重叠扩增PCR技术将鸡堆形艾美球虫子孢子表面抗原3-1E基因片段(Ea3-1E)与编码鸡白细胞介素15成熟蛋白基因片段(mChIL-15)进行串联连接,并在这2个基因片段之间引入4个柔性连接肽SPGS,获得融合基因Ea3-1E-linker-mChIL-15。以pcDNA3.1(+)为载体构建并鉴定真核表达质粒pcD-NA3.1-Ea3-1E-linker-mChIL-15。应用磷酸钙法将质粒体外转染293T细胞,通过间接免疫荧光技术和免疫组织化学技术对重组质粒的体外瞬时表达进行检测。将重组质粒pcDNA3.1-Ea3-1E-linker-mChIL-15、pcDNA3.1-Ea3-1E和pcDNA3.1分别于14和21日龄经腿部肌肉注射免疫SPF雏鸡,28日龄时,除非免疫非感染组外,各组感染5×104个堆形艾美球虫孢子化卵囊,观察真核表达质粒的免疫保护效果。结果显示,成功构建了融合基因3-1E-linker-mChIL-15,转染后第30小时可检测到融合基因编码蛋白在293T细胞中的瞬时表达。与质粒pcDNA3.1-Ea3-1E相比,质粒pcDNA3.1-Ea3-1E-linker-mChIL-15二次免疫后可提供明显的抗球虫免疫保护效果,提高相对增重率(96.48%),降低卵囊排出率(68.35%),降低十二指肠病变记分等。抗球虫指数为183.78。     

小反刍兽疫病毒F和H蛋白在CHO细胞中的融合表达及其免疫原性的研究

以小反刍兽疫病毒(peste des petits ruminants virus,PPRV)疫苗株Nigeria75/1基因序列为基础,构建了融合表达PPRV囊膜蛋白F和H胞外区的真核重组质粒pcDNA3.1-F-H,在悬浮CHO细胞中进行分泌表达。离心收集转染后的细胞培养上清进行亲和层析纯化,纯化样品经Western-blot鉴定,融合蛋白与PPRV阳性血清和His标签抗体都能发生免疫反应。使用纯化后的融合蛋白免疫BALB/c小鼠,可诱导小鼠体内产生针对PPRV的特异性抗体。上述研究结果为小反刍兽疫新型亚单位疫苗的研制奠定了基础。     

鸡堆形艾美球虫3-1E基因真核表达质粒的构建及其抗球虫免疫保护作用

应用反转录-聚合酶链式反应(RT-PCR)技术从堆形艾美球虫SH株子孢子中扩增3-1E基因片段,将其克隆入真核表达载体pcDNA3.1(+)中,构建重组表达质粒pcDNA3.1(+)-3-1E.质粒纯化后体外转染293T细胞,通过间接免疫荧光技术和免疫组织化学技术检测3-1E蛋白在转染细胞中的表达情况.将构建的重组质粒以及空载体质粒分别于14、21日龄分2次经腿部肌肉注射免疫SPF雏鸡,28日龄除非免疫非感染对照组外各组攻击性感染5×104个堆形艾美球虫孢子化卵囊,观察真核表达质粒对球虫感染鸡的免疫保护作用并探讨其免疫保护机理.结果显示,与载体对照组相比,质粒pcDNA3.1(+)-3-1E 2次免疫后可显著提高脾CD8 T淋巴细胞亚型数量,并具有一定的抗球虫免疫保护作用,可显著减少每克粪便的卵囊排出量,降低十二指肠病变记分,减少增重下降等.     

鸡IL-18基因重组真核表达载体的构建及其表达产物的生物学活性

从pMDChIL18克隆质粒扩增获得了ChIL18全基因片段,并将其重组到真核表达载体pcDNA3.1( )。经酶切、质粒PCR鉴定和基因测序,鸡IL18全基因被正确重组到pcDNA3.1( )真核表达质粒上;将重组真核表达质粒pcDNA3.1( )ChIL18转染COS7细胞,转染细胞中含ChIL18基因的mRNA。SDSPAGE分析表明,表达产物是与ChIL18相符的约23ku的蛋白条带。鸡淋巴细胞转化试验表明,表达产物对鸡淋巴细胞具有明显诱导转化作用。     

Terrorism in Jammu and Kashmir in theory and practice

This article argues there is a need for a more nuanced analysis of terrorism in Jammu and Kashmir than has been available so far. First, the popular legitimacy of violent groups has little bearing on their operation. Rather, the keys to the intensity of terrorist activity are held by Pakistan's military establishment. Second, the supposedly secular-nationalist movement of the early 1990s was in fact deeply Islamist in character; there has been a greater unity of thought underpinning terrorism than the literature admits. Finally, the article argues, the operation of terrorism needs to be read not simply in the limited context of Jammu and Kashmir, but as part of a larger South Asian crisis of identity.     

Institutional divergence and convergence in the Asia-Pacific? ASEAN in practice and in theory

This paper gives attention to the geopolitics related to the Association of Southeast Asian Nations (ASEAN) as an alternative model of regionalism in theory and practice. Offering a rough periodization of ASEAN in IR theory, it considers interacting theoretical and empirical developments, and their geopolitics as one way to think about, first, ASEAN, its defining dynamics and processes of change, and, second, ASEAN's relationship to a larger IR theory literature defined by US preoccupations and the institutional trajectory of the European Union. Three periods are considered: a Cold War period, when ASEAN norms and practices developed relatively insulated from great power expectations and theorizing about ASEAN was minimal; the 1990s, when constructivist theorizing encouraged new thinking about alternative institutional models; and the 2000s, a period characterized by correlating great power pressures and a ‘functional’ turn in academic and theoretical debates about ASEAN. Special attention is given to the United States as a major, leading actor in both world politics and the institutionalization of international relations as a discipline, as well as the possibilities and constraints of institutional divergence in theory and practice.     

Islam and politics in Europe and in Asia: Comparative reflections

This article compares the role of religion, and of Islam in particular, in politics in Europe and in South and South East Asia. It starts out with the policy dilemmas facing France, Europe's most secular country that also has Europe's largest immigrant Muslim community. After long debates nation-wide Muslim organisation is now sponsored by the state in order to strengthen moderate Islam in France. In contrast, explicit Christian parties are in decline in most of Europe. Those who are still electorally successful are Christian mostly in name only and have turned into centre-right conservative people's parties instead. Religious discourse in politics has hence vanished almost entirely in Europe. In difference in Asia Islamic opposition parties have managed to set increasingly the political agenda in the majority Islamic states. In those countries with an Islamic minority their public religious agitation serves to strengthen their ethnic minority identity. Meaningful bi-continental dialogue needs to be aware of this discrepancy in religious politics.     

Simultaneous in Growth and Different in Problems

Lu: On many occasions, politics and economy are in the same strain. Economic life is the only process that runs through history and undergoes environmental changes. World economy is the only world system. In today's world, the closer ties among various countries in e- conomy cause the complexity of political and foreign affairs. Last year's world economy took on a new feature. According to past experiences, when the economy in a region turns better, problems will usually arise in another regi…     

经济继续衰退 改革面临困境——2002年拉美经济形势述评

20 0 2年 ,拉丁美洲和加勒比地区 (以下简称拉美地区 )的经济形势可以概括为 :经济继续衰退 ,改革面临困境。根据联合国拉美经济委员会最近发表的年度总结报告 ,拉美地区经济在上年仅增长 0 .4%的基础上 ,2 0 0 2年出现负增长 ( -0 .5% ) ,为最近 2 0年来首次出现负增长。人均国内生产总值 ( GDP)为 -1 .9%。阿根廷、乌拉圭和委内瑞拉的 GDP分别下降 1 1 %、1 0 .5%和 7% ,是衰退最严重的国家 ;秘鲁、多米尼加和厄瓜多尔 3国的增长率超过3 % ,算是拉美地区的佼佼者 ;其他国家的增长率都不到 3 % ,其中巴西和墨西哥两个地区大国分别只增长…     

中印关系的回顾与展望

60年来,中印两国关系走过了一个不平坦的过程,大体上经历了"蜜月"期、冲突期、冷战期、解冻期、回暖期和加热期这样6个阶段。历史的经验教训说明,中印两国合则互利,斗则两伤,这已成为中印双方的共识。人们有理由相信,随着双方政治互信、经贸互惠、文化互通的日益加强,中印两国能够创造出共同繁荣的景象,能够为亚洲乃至世界和平作出重要贡献。     

明清小说《剪灯新话》在朝鲜和越南的传播

本文主要探讨明清小说《剪灯新话》在朝鲜的传播、影响及其本土化，并与《剪灯新话》在越南的传播进行宏观对比，探讨《剪灯新话》在朝鲜和越南的不同境遇。     

中日两国对东南亚基础设施投资的竞合及影响研究

在21世纪第二个十年,中国与日本在对东南亚基础设施投资方面展开激烈竞争和初步合作.相对而言,在甄选东南亚基础设施项目进行投资过程中,中国更加追求地缘政治目标,日本则凸显了浓重的重商主义色彩,这在某种程度上缓解了双方的直接竞争.在对东南亚基础设施投资中,中国更多地运用了一种政府驱动、需求引导、自上而下的方法,日本则主要采取了市场驱动、私营部门大力参与、自下而上的方法,这瓦解了双方合作的基础.目前,中国与日本对东南亚基础设施投资的合作只在第三方市场少量地展开.中日两国在东南亚基础设施投资方面的竞争是双方争夺地区影响力的一部分.但是,中国与日本在东南亚各国的基础设施投资竞争并不会造成东盟内部的分裂,相反,考虑到基础设施建设的非流动性,这必将造福于东南亚人民.     

Party competition in emerging democracies: representation and effectiveness in post-communism and beyond

Competition among political parties is subject to two demands: representation and effectiveness. This poses a dilemma for democratization, as the political opening creates pressures for the representation of long-suppressed voices, but the strain of socio-economic transformation engenders pressures for greater effectiveness in building market economies and democratic polities. How do new democracies cope with this dilemma? This article extends the author's previously published work on party-system institutionalization by focusing explicitly on this problem, including on how electoral reforms affect the distinct pulls of representation and effectiveness. Based on data from eastern European and former Soviet states compared to western European and Latin American experiences during their initial periods of democratization, the evidence shows that the institutionalization of representation and effectiveness in post-communism is more hazardous. Politics in the post-communist region is characterized by numerous contending parties, weak political actors and floating constituencies. Volatility is not only higher in comparison to other regions but continues unabated during successive elections. These conditions contribute to the ‘ineffective representation’ evident in the relatively large share of wasted votes at each election, with significant sectors of the electorate left out from legislative representation. Electoral reforms reflect the twin pressures: changes in formula in the direction of proportional representation favour broader representation, but higher thresholds seeking greater effectiveness make more difficult entry into parliament. The resulting mechanical and strategic effects confirm the expected direction of the reforms, although the contrary pulls towards representation and effectiveness render difficult the stabilization of party competition.